Producing high-affinity antibodies that potently neutralize target pathogens remains a central goal for vaccine research. Many promising vaccine antigens fail to elicit long-lasting protective antibodies due to inefficient generation of high-affinity memory B cells. While memory B cell development is an active area of research, major gaps remain in our understanding of cognate regulation by CD4+ TH cells and the rules governing ongoing evolution of antigen-specific B cell memory. Research Focus: In these recent studies, we demonstrate that all memory B cell responses require antigen-specific mTH cells. Importantly, direct in situ labeling revealed both Bcl-6+ and Blimp-1+ CD4+ TH cells in the follicular regions of reactive LNs after recall. In support of this observation, using single cell T-qPCR, we demonstrate a transcriptional divide between two tetramer-binding CXCR5+ mTFH compartments with differential expression of Bcl-6 and Prdm-1 at recall. Based on these findings, we propose a division in cognate regulatory function with separable mechanisms used by the different mTFH compartments that may also pre-exist at initial priming. We predict that this division of cognate regulatory function can be targeted to control, remodel and re-assort antigen-specific memory B cell fate at antigen recall with high impact on durable immune protection. Specific Aims: We have developed multi-dimensional single cell strategies to track complex cellular behaviors in vivo and can temporally delete of these two major transcriptional repressors in a cell- specific manner using mixed BM chimeras. We will use this approach: SA-1: to study the molecular control of antigen-specific mTFH differentiation. SA-2: to dissect and modify the mechanisms and GC-regulating function of Bcl-6+ mTFH cells SA-3: to examine the cognate regulatory mechanisms used by Prdm-1+ mTFH cells. Impact: We use polyclonal murine models of protein vaccination with state-of-the-art single cell analyses of antigen-specific immune function. These high-resolution studies of model antigens have the capacity to shift the basic conceptual framework that surrounds existing vaccine paradigms. Importantly, these basic new principles and assays can be used to re-design contemporary vaccine formulations that optimize high-affinity B cell immunity to more complex antigens.